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In freshwater wetlands, competitive and cooperative interactions between respiratory, fermentative and methanogenic microbes mediate the decomposition of organic matter. These interactions may be disrupted by saltwater intrusion disturbances that enhance the activity of sulfate-reducing bacteria (SRB), intensifying their competition with syntrophic bacteria and methanogens for electron donors. We simulated saltwater intrusion into wetland soil microcosms and examined biogeochemical and microbial responses, employing metabolic inhibitors to isolate the activity of various microbial functional groups. Sulfate additions increased total carbon dioxide production but decreased methane production. Butyrate degradation assays showed continued (but lower) levels of syntrophic metabolism despite strong demand by SRB for this key intermediate decomposition product and a shift in the methanogen community toward acetoclastic members. One month after removing SRB competition, total methane production recovered by only ∼50%. Similarly, butyrate assays showed an altered accumulation of products (including less methane), although overall rates of syntrophic butyrate breakdown largely recovered. These effects illustrate that changes in carbon mineralization following saltwater intrusion are driven by more than the oft-cited competition between SRB and methanogens for shared electron donors. Thus, the impacts of disturbances on wetland biogeochemistry are likely to persist until cooperative and competitive microbial metabolic interactions can recover fully., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up to 98% assignment accuracy at the species level. The observed community proportions for “equal” and “rare” synthetic libraries were close to the known proportions, deviating from 0.1% to 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at <0.05% of DNA in the total mixture), 91% of reads were assigned to the correct species, 93% of reads were assigned to the correct genus, and >99% of reads were assigned to the correct family. Conclusions: At the current level of output and sequence quality (just under 4 × 103 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high-quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities., (© The Author 2017. Published by Oxford University Press.)

To better understand the distribution of soil microbial communities at multiple spatial scales, a survey was conducted to examine the spatial organization of community structure in a wheat field in eastern Virginia (USA). Nearly 200 soil samples were collected at a variety of separation distances ranging from 2.5 cm to 11 m. Whole-community DNA was extracted from each sample, and community structure was compared using amplified fragment length polymorphism (AFLP) DNA fingerprinting. Relative similarity was calculated between each pair of samples and compared using geostatistical variogram analysis to study autocorrelation as a function of separation distance. Spatial autocorrelation was found at scales ranging from 30 cm to more than 6 m, depending on the sampling extent considered. In some locations, up to four different correlation length scales were detected. The presence of nested scales of variability suggests that the environmental factors regulating the development of the communities in this soil may operate at different scales. Kriging was used to generate maps of the spatial organization of communities across the plot, and the results demonstrated that bacterial distributions can be highly structured, even within a habitat that appears relatively homogeneous at the plot and field scale. Different subsets of the microbial community were distributed differently across the plot, and this is thought to be due to the variable response of individual populations to spatial heterogeneity associated with soil properties., (c2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.)

Small-scale variations in bacterial abundance and community structure were examined in salt marsh sediments from Virginia's eastern shore. Samples were collected at 5 cm intervals (horizontally) along a 50 cm elevation gradient, over a 215 cm horizontal transect. For each sample, bacterial abundance was determined using acridine orange direct counts and community structure was analyzed using randomly amplified polymorphic DNA fingerprinting of whole-community DNA extracts. A geostatistical analysis was used to determine the degree of spatial autocorrelation among the samples, for each variable and each direction (horizontal and vertical). The proportion of variance in bacterial abundance that could be accounted for by the spatial model was quite high (vertical: 60%, horizontal: 73%); significant autocorrelation was found among samples separated by 25 cm in the vertical direction and up to 115 cm horizontally. In contrast, most of the variability in community structure was not accounted for by simply considering the spatial separation of samples (vertical: 11%, horizontal: 22%), and must reflect variability from other parameters (e.g., variation at other spatial scales, experimental error, or environmental heterogeneity). Microbial community patch size based upon overall similarity in community structure varied between 17 cm (vertical) and 35 cm (horizontal). Overall, variability due to horizontal position (distance from the creek bank) was much smaller than that due to vertical position (elevation) for both community properties assayed. This suggests that processes more correlated with elevation (e.g., drainage and redox potential) vary at a smaller scale (therefore producing smaller patch sizes) than processes controlled by distance from the creek bank., (c2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.)

The effects of liposome-encapsulated diethylstilbestrol (DES) on the size of the ventral prostate gland and total testosterone levels in rats were studied. DES was encapsulated in phosphatidycholine-cholesterol-stearylamine-alpha-tocopherol containing multilamellar liposomes in a molar ratio of 4:5:1:0.01. The liposome suspension or free DES plus blank liposomes were administered sc on days 1, 3, and 5 at a dose of 400, 87, or 12 micrograms/kg. Plasma testosterone values showed a decrease with dose for animals treated with either entrapped DES or free DES. Prostate gland weight per g animal and prostatic protein mg per g animal on day 7 were significantly decreased in the treated animals compared to those in controls dosed with DES plus buffer containing liposomes. In addition, there was a body weight loss for the high dose DES-encapsulated animals and for high and medium dose DES plus blank liposome-treated and blank liposome-treated animals over the treatment period, although only the DES-encapsulated group value was statistically significant (P less than 0.05). Animals at the other doses did not lose weight, but exhibited weight gains which were less than those observed for sham-injected animals. Rats that received 7 micrograms/kg liposome-entrapped DES for 4 weeks had significantly lower prostate gland weights, prostatic protein levels, and testosterone levels than control animals receiving the same regimen of free DES plus blank liposomes. Results from the studies indicate that liposome-entrapped DES was more effective in decreasing the size of the prostate gland and that the desired therapeutic end point (prostate ablation) may be attained with a lower dose of DES, which would decrease the risk of weight loss in the experimental animals.

Constant intravenous infusion (65 to 260 ng/min) of prostaglandin E2 (PGE2) produced an elevation of the plasma calcium concentration in unanesthetized rats in a period of 4 h. The increase in plasma calcium was related to the amount of PGE2 infused, and the hypercalcemia persisted for at least 10 h during continuous infusion. There was also a small increase in plasma inorganic phosphorus concentration. The increase in plasma calcium was not due to hemoconcentration; in fact, there was a small decrease in hematocrit during infusion of PGE2, and no change in total plasma protein concentration was detected. Infusion of PGF2, did not raise plasma calcium concentration. We conclude that under appropriate conditions, the exogenous administration of PGE2 can cause an elevation of plasma calcium in the intact rat.